Project Summary RNA plays a diverse set of roles in biology. Localization of RNA in different regions of the cell establishes the developmental program of multicellular organisms and errors in this process have been implicated in neurological disorders. Long non-coding RNAs and microRNAs provide sophisticated regulatory functions that are still being elucidated. Cell-to-cell variations in RNA expression have been identified as a mechanism through which cancer cells become drug tolerant. Despite the profound influence of RNA, it remains a considerable challenge to monitor in living cells the expression dynamics and localization of multiple RNAs. Existing techniques have thus far suffered from substantial drawbacks, such as inefficient delivery, high background fluorescence, limited multiplexing, or require sequence modifications or labelling of the RNA being imaged. This proposal describes an innovative strategy for multiplexed imaging of RNAs in live cells. This strategy employs genetically encoded RNA-based probes termed molecular fuses that undergo an intramolecular chain reaction upon binding to a target RNA that mimics the propagation of a flame along a fuse. These self-assembling systems will enable direct imaging of RNAs down to the single-copy level and visualization of RNA expression in heterogeneous cell populations. Key to this imaging strategy is a new approach to triggered self-assembly in which a domino-like chain reaction is initiated within a single molecule in response to an RNA binding event. This unimolecular self-assembly paradigm stands in stark contrast to the prevailing approach that relies on interactions between multiple short probes whose reaction kinetics are limited by diffusion in the cytoplasm. The immediate benefits of this single-molecule self-assembly strategy are threefold. First, encoding the self- assembly program within a single RNA leads to an enhanced local concentration of interacting domains and dramatically increases reaction rates. Second, the high reaction kinetics and strong thermodynamics driving the intramolecular self-assembly reaction lead to high assembly yields. Third, programming of self-assembly within a single strand of RNA leads to triggered probes with a precisely defined number of fluorescent labeling sites for each target RNA enabling highly multiplexed imaging capabilities. To realize this innovative live-cell RNA imaging platform, we will accomplish four principal aims: (1) Construct RNA sensors that bind to target RNAs and output aptamers with different fluorescent ligands. (2) Develop molecular fuse RNAs that undergo intramolecular chain reactions with RNAs in the cell. (3) Implement multicolor molecular fuses with combinations of aptamers for multiplexed RNA imaging. (4) Generate molecular fuses that activate dozens of aptamers for single-molecule RNA imaging. The proposed research will enable us to visualize RNA expression and localization in a deeper and less perturbative fashion than has ever been possible before. It will also provide a new strategy for triggered nucleic acid self-assembly with profound implications for medicine, biotechnology, and nanotechnology.